Tetanus toxin fragment C expressed in live Salmonella vaccines enhances antibody responses to its fusion partner Schistosoma haematobium glutathione S-transferase

Tetanus toxoid has been used widely as an adjuvant. The atoxic fragment C from tetanus toxin (TetC) is potently immunogenic when expressed in Salmonella vaccine strains and has been used as a fusion partner for antigens (Ag). However, there has been no formal comparison of the immunomodulatory impact of TetC on its fusion partners. In this study, we have addressed this important issue. The protective 28-kDa glutathione S-transferase (GST) from Schistosoma haematobium (Sh28GST) was expressed either as a fusion to TetC or as the full-length Sh28GST alone in a nonvirulent aroA-attenuated strain of Salmonella enterica serovar Typhimurium. The Sh28GST proteins were soluble and stably expressed in Salmonella, as evaluated by Western blotting with TetC and/or Sh28GST antisera. Mice were immunized orally with a single dose of the live recombinant Salmonella. The constructs were stable in mice but, dramatically, only the strain expressing the TetC-Sh28GST fusion elicited significant antibody (Ab) responses directed against Sh28GST as determined by enzyme-linked immunosorbent assay. An analysis of the isotype profiles showed that these mice also produced anti-Sh28GST immunoglobulin A and GST-neutralizing assays revealed high levels of neutralizing Abs in sera. These are important correlates of protection in schistosomiasis. In addition, stimulation of spleen cells from immunized mice with Sh28GST Ag showed that both strains, expressing Sh28GST alone or the TetC-Sh28GST fusion, were able to stimulate the secretion of Th1-related cytokines (gamma interferon and interleukin 2) to comparable levels. Thus, TetC has modulated the immune responses generated against its fusion partner, Sh28GST, by markedly enhancing the Ab responses elicited. These results have important implications in the rational development of live vaccines.     

Evaluation of a rapid enzyme-linked immunosorbent assay for IgG antibodies to Aspergillus fumigatus in the serological diagnosis of allergic aspergillosis

A range of 6 somatic and culture filtrate antigens of Aspergillus fumigatus were evaluated in a rapid ELISA procedure for anti-A. fumigatus IgG where the component incubation times had been reduced to 10 min. Sera from patients with allergic aspergillosis, patients with suspected allergic aspergillosis, and asthmatic patients with or without A. fumigatus precipitins were tested. For all antigens, levels of anti-A. fumigatus IgG were higher in patients with allergic aspergillosis than in the other 3 groups. Low levels of specific IgG were, however, detected in asthmatic patients who had no precipitins against A. fumigatus. None of the antigen preparations enabled all patients with proven or suspected allergic aspergillosis to be separated from the other 2 groups of asthmatic patients. Positive-negative discrimination in ELISA was achieved by the inclusion of 10 pools of precipitin test-negative sera from the 50 asthmatics without A. fumigatus precipitins. The number of sera that were classed as positive in ELISA ranged from 9 to 15 in the allergic aspergillosis group, depending on the antigen used; in the suspected aspergillosis group, the number of positive reactions ranged from 1 to 8, while in the asthmatics with precipitins, the number ranged from 0 to 2.     

Novel type of fimbriae encoded by the large plasmid of sorbitol-fermenting enterohemorrhagic Escherichia coli O157:H(-)

Sorbitol-fermenting (SF) enterohemorrhagic Escherichia coli (EHEC) O157:H(-) have emerged as important causes of diarrheal diseases and the hemolytic-uremic syndrome in Germany. In this study, we characterized a 32-kb fragment of the plasmid of SF EHEC O157:H(-), pSFO157, which differs markedly from plasmid pO157 of classical non-sorbitol-fermenting EHEC O157:H7. We found a cluster of six genes, termed sfpA, sfpH, sfpC, sfpD, sfpJ, and sfpG, which mediate mannose-resistant hemagglutination and the expression of fimbriae. sfp genes are similar to the pap genes, encoding P-fimbriae of uropathogenic E. coli, but the sfp cluster lacks homologues of genes encoding subunits of a tip fibrillum as well as regulatory genes. The major pilin, SfpA, despite its similarity to PapA, does not cluster together with known PapA alleles in a phylogenetic tree but is structurally related to the PmpA pilin of Proteus mirabilis. The putative adhesin gene sfpG, responsible for the hemagglutination phenotype, shows significant homology neither to papG nor to other known sequences. Sfp fimbriae are 3 to 5 nm in diameter, in contrast to P-fimbriae, which are 7 nm in diameter. PCR analyses showed that the sfp gene cluster is a characteristic of SF EHEC O157:H(-) strains and is not present in other EHEC isolates, diarrheagenic E. coli, or other Enterobacteriaceae. The sfp gene cluster is flanked by two blocks of insertion sequences and an origin of plasmid replication, indicating that horizontal gene transfer may have contributed to the presence of Sfp fimbriae in SF EHEC O157:H(-).     

Effects of fluorodeoxyuridine on the developing inner ear of the rat

The inner ear in rats develops from the surface ectoderm on day 8 of a 22-day gestational period. Labeled thymidine incorporation studies have indicated that in the developing inner ear most of the cells undergo terminal mitosis between gestational days 13 and 15. During this period the developing inner ear would be particularly vulnerable to environmental hazards. To test this hypothesis, pregnant rats were given a single intraperitoneal injection of 5-fluoro-2'-deoxyuridine (FUdR), an antimitotic substance, on gestational days 12 to 16. The rats also received one injection of 3H-thymidine 1 h prior to the removal of the fetuses. The animals were killed after various time intervals following the treatment, and the otocysts or inner ears were prepared for morphologic observations and biochemical assays. The cells in the inner ear of rats exposed to FUdR exhibited pyknotic nuclei and chromatolytic degeneration, and they eventually died. By 4 h after the administration of FUdR, pyknotic nuclei were seen in the antiluminal zone of the otic epithelium, and there was a substantial decrease in the number of the otic cells. This decline in cell number was seen until 24 h after treatment. However, the inner ears from the fetuses exposed to FUdR during gestational days 12--15 showed complete recovery from the toxic effects of the drug when examined on day 21 of gestation. The phenomenon of programmed cell death observed in the developing inner ear of the rat indicates that more cells are produced during the earlier stages of development than are required for the definitive adult structures. This phenomenon may represent an important protective feature. The redundant production of cells perhaps allows the developing otocysts to respond to an environmental stress by subtotal destruction of cells from the pool of undifferentiated cells, resulting in relatively fewer congenital anomalies of the inner ear.     

先天性软骨发育不全成纤维细胞生长因子受体3基因1138位核苷酸点突变的检测

目的 验证成纤维细胞生长因子受体3(fibroblast growth factor receptor 3，FGFR3)跨膜区1138位核苷酸为先天性软骨发育不全突变热点及用变性梯度电泳方法筛查的效果。方法 对17例临床诊断为先天性软骨发育不全患者进行基因组DNA聚合酶链反应－限制性酶切片段长度多态性（polymerase chain reaction-restriction fragment length polymorphism,PCR-RFLP)分析，并用变性梯度凝胶电泳（denaturing gradient gel electrophoresis,DGGE)进行其它突变位点的筛查。结果 17例患者中14例RFLP检测显示能被Sfc I酶切，提示存在1138位核苷酸G→A的转换，且均为杂合子。17例用Msp I酶切均阴性，提示无G→C的颠换。DGGE方法的筛查中，酶切阳性的14例标本均显示存在杂合型突变。3例PCR－RFLP检测阴性的标本未显示突变位点，提示在该扩增区域确实不存在突变位点。结论 FGFR3跨膜区1138核苷酸G→A的突变是软骨发育不全的主要发病机理，DGGE可作为筛查某一区域基因突变的敏感而可靠的检测手段，尤其是对杂合子的检测。     

Evidence that prostate gonadotropin-releasing hormone receptors mediate an anti-tumourigenic response to analogue therapy in hormone refractory prostate cancer

Gonadotropin-releasing hormone analogue (GnRHa) therapy is an established method of androgen withdrawal in the treatment of prostate cancer. The present study investigated if the expression of prostate GnRH receptors (GnRHRs) might influence the response to GnRHa. GnRHR protein expression was first studied in a panel of prostate cancer cell lines. In androgen-dependent cells, GnRHR expression was unchanged following acute or chronic androgen withdrawal. In these cells, GnRHa significantly inhibited androgen-induced cell proliferation (p = 0.01). In contrast, GnRHa was unable to further suppress basal levels of cell proliferation induced by androgen withdrawal. In androgen-independent prostate cancer cells, variable levels of GnRHR expression were observed. In these cells, GnRHa treatment blocked cell proliferation (p = 0.001) and invasion (up to 70%) induced by fibroblast growth factor stimulation. Crucially, this effect was only evident in cells that expressed high levels of the GnRHR. GnRHa treatment also significantly inhibited the ability of these cells to recover from a cytotoxic insult (50% inhibition). The clinical significance of prostate GnRHR was tested by immunohistochemistry in a preliminary cohort of patients treated with GnRHa or surgical castration. There was no association between GnRHR expression and pathological grade, clinical stage, time to PSA nadir (p = 0.82) (n = 35) or progression to hormone refractory disease (p = 0.22) (n = 21), irrespective of the treatment method. GnRHa therapy in the presence of high GnRHR expression however, was found to be associated with longer disease-specific survival (mean survival 85 months, p = 0.002). In contrast, high GnRHR expression was not associated with survival among surgically castrated patients (mean survival 50 months, p = 0.7). Taken together, these data support the notion of a functional interaction between GnRHa and the GnRHR, which results in an anti-tumourigenic effect on prostate cancer cells. Findings from this report have direct implications for the use of GnRHR as a novel therapeutic target in hormone refractory prostate cancer.     

A phase I study of dexosome immunotherapy in patients with advanced non-small cell lung cancer

BACKGROUND: There is a continued need to develop more effective cancer immunotherapy strategies. Exosomes, cell-derived lipid vesicles that express high levels of a narrow spectrum of cell proteins represent a novel platform for delivering high levels of antigen in conjunction with costimulatory molecules. We performed this study to test the safety, feasibility and efficacy of autologous dendritic cell (DC)-derived exosomes (DEX) loaded with the MAGE tumor antigens in patients with non-small cell lung cancer (NSCLC). METHODS: This Phase I study enrolled HLA A2+ patients with pre-treated Stage IIIb (N = 4) and IV (N = 9) NSCLC with tumor expression of MAGE-A3 or A4. Patients underwent leukapheresis to generate DC from which DEX were produced and loaded with MAGE-A3, -A4, -A10, and MAGE-3DPO4 peptides. Patients received 4 doses of DEX at weekly intervals. RESULTS: Thirteen patients were enrolled and 9 completed therapy. Three formulations of DEX were evaluated; all were well tolerated with only grade 1-2 adverse events related to the use of DEX (injection site reactions (N = 8), flu like illness (N = 1), and peripheral arm pain (N = 1)). The time from the first dose of DEX until disease progression was 30 to 429+ days. Three patients had disease progression before the first DEX dose. Survival of patients after the first DEX dose was 52-665+ days. DTH reactivity against MAGE peptides was detected in 3/9 patients. Immune responses were detected in patients as follows: MAGE-specific T cell responses in 1/3, increased NK lytic activity in 2/4. CONCLUSION: Production of the DEX vaccine was feasible and DEX therapy was well tolerated in patients with advanced NSCLC. Some patients experienced long term stability of disease and activation of immune effectors.     

Boleodorus similis n. sp. (Nematoda: Nothotylenchinae) from India

Summary A new species, B. similis, is described from male and female specimens collected from soil around roots of Plumeria acutifolia Poir from North India. It is distinguished by a short and robust body, a broad lateral field, a more posteriorly located vulva in female and the position of phasmid and the posterior extension of lateral field in male.With 8 Figures in the Text     

Anatomical variations in human carotid bodies.

The variations in anatomical structure and position of both carotid bodies were noted in 100 consecutive subjects who came to necropsy. Considerable variations in form were found. Although most carotid bodies (83% on the right and 86% on the left) were of the classic ovoid type, an appreciable minority was bilobed (9% on the right and 7% on the left) or double (7% on the right and 6% on the left); 1% were leaf shaped. All these anatomical variants have to be distinguished from the pathologically enlarged carotid body that may have a smooth or finely nodular surface. Anatomical variants (such as the bilobed) may themselves enlarge as a consequence of carotid body hyperplasia.